The eukaryotic origin recognition complex (ORC) selects the genomic sites where pre-replication complexes are assembled and DNA replication begins. In proliferating mammalian cells, ORC activity appears to be regulated by reducing the affinity of the Orc1 subunit for chromatin during S-phase, and then preventing reformation of a stable ORC/chromatin complex until mitosis is completed, and a nuclear membrane is assembled. Here we show that part of the mechanism by which this is accomplished is through the selective association of Orc1 with Cdk1(Cdc2)/cyclin A during the G2/M?phase of cell division. This association accounted for the appearance of hyperphosphorylated Orc1 in M-phase cells that was subsequently dephosphorylated during the M to G1 transition. Moreover, inhibition of Cdk activity in metaphase cells resulted in rapid binding of Orc1 to chromatin. However, chromatin binding was not mediated through increased affinity of Orc1 for Orc2, suggesting that additional events are involved in the assembly of functional ORC/chromatin sites. These results reveal that the same cyclin-dependent protein kinase that initiates mitosis in mammalian cells also concomitantly inhibits assembly of functional ORC/chromatin sites. Previous studies have suggested that origin recognition complex (ORC) activity in mammalian cells is regulated by cell cycle dependent changes in its largest subunit, Orc1. Here we demonstrate that the same events that modify Orc1 behavior during cell proliferation also prevent it from inducing apoptosis. Ectopic expression of Orc1 at levels equivalent to endogenous Orc1 rapidly induced p53-independent apoptosis in the absence of endoreduplication, as evidenced by induction of caspase-3 activity, changes in cell structure, perinuclear accumulation of Orc1, DNA fragmentation, and loss of cell adhesion. Remarkably, Orc2 was unique among the ORC subunits in that it did not induce apoptosis. Moreover, coexpression of Orc2 with Orc1 suppressed apoptosis, suggesting a critical role for Orc2 in ORC assembly. In addition, either a single ubiquitin adduct, or substitution of aspartic acid at multiple Cdk phosphorylation sites suppressed Orc1 induction of apoptosis and caused Orc1 to accumulate in the cytoplasm. Thus, either the mono- or poly-ubiquitination of Orc1 that occurs during S-phase, and the hyperphosphorylation of Orc1 that occurs during G2/M-phase can prevent assembly of functional ORC/chromatin sites. Furthermore, failure to carry out these events can result in apoptosis.